Duplication of functional polyadenylation signals in polyomavirus DNA does not alter efficiency of polyadenylation or transcription termination.

We constructed viable insertion mutants of polyomavirus that contain duplications of the nucleotide sequences surrounding the polyadenylation sites for both E- and L-strand RNAs. Our results showed that formation of poly(A)+ 3'termini of L-strand mRNAs requires sequence elements located between 12 and 87 nucleotides downstream of AAUAAA. No more than 19 nucleotides upstream and 44 nucleotides downstream of AAUAAA are required for polyadenylation of E-strand mRNAs. Our results and those of others suggest that there are three distinct sequence elements required for mRNA 3' end formation: AAUAAA and two downstream elements. An insertion mutant containing two adjacent functional polyadenylation signals produced E-strand and L-strand mRNAs with 3' ends at both sites. However, the overall level of polyadenylation of L-strand RNAs was not increased over the low (10 to 25%) levels seen with wild-type virus. Neither was the efficiency of termination of L-strand transcription increased in mutant virus-infected cells. We conclude that factors required for both polyadenylation and transcription termination are limiting in polyomavirus-infected mouse cells.